Comparative analysis of homologous aminopeptidase PepN from pathogenic and non-pathogenic mycobacteria reveals divergent traits

Mycobacterium tuberculosis (Mtb) secretes proteases and peptidases to subjugate its host. Out of its sixty plus proteases, atleast three are reported to reach host macrophages. In this study, we show that Mtb also delivers a lysyl alanine aminopeptidase, PepN (Rv2467) into host macrophage cytosol. Our comparative in silico analysis shows PepNMtb highly conserved across all pathogenic mycobacteria. Non-pathogenic mycobacteria including M. smegmatis (Msm) also encode pepN. PepN protein levels in both Mtb (pathogenic) and Msm (non-pathogenic) remain uniform across all in vitro growth phases. Despite such tight maintenance of PepNs’ steady state levels, upon supplementation, Mtb alone allows accumulation of any excessive PepN. In contrast, Msm does not. It not only proteolyzes, but also secretes out the excessive PepN, be it native or foreign. Interestingly, while PepNMtb is required for modulating virulence in vivo, PepNMsm is essential for Msm growth in vitro. Despite such essentiality difference, both PepNMtb and PepNMsm harbor almost identical N-terminal M1-type peptidase domains that significantly align in their amino acid sequences and overlap in their secondary structures. Their C-terminal ERAP1_C-like domains however align much more moderately. Our in vitro macrophage-based infection experiments with MtbΔpepN-expressing pepNMsm reveals PepNMsm also retaining the ability to reach host cytosol. Lastly, but notably, we determined the PepNMtb and PepNMsm interactomes and found them to barely coincide. While PepNMtb chiefly interacts with Mtb’s secreted proteins, PepNMsm primarily coimmunoprecipitates with Msm’s housekeeping proteins. Thus, despite high sequence homology and several common properties, our comparative analytical study reveals host-centric traits of pathogenic and bacterial-centric traits of non-pathogenic PepNs.结核分枝杆菌（Mycobacterium tuberculosis, Mtb）会分泌蛋白酶与肽酶以操控宿主。该菌的六十余种蛋白酶中，已有至少三种被证实可抵达宿主巨噬细胞。本研究证实，Mtb还可将赖氨酰丙氨酸氨基肽酶（lysyl alanine aminopeptidase）PepN（Rv2467）递送至宿主巨噬细胞胞质内。我们通过对比生物信息学（in silico）分析发现，结核分枝杆菌来源的PepN（PepN_Mtb）在所有致病性分枝杆菌中均高度保守。包括耻垢分枝杆菌（Mycobacterium smegmatis, Msm）在内的非致病性分枝杆菌，同样编码pepN基因。无论是致病性的Mtb还是非致病性的Msm，其PepN的蛋白水平在所有体外（in vitro）培养的生长阶段均保持恒定。尽管二者均严格维持PepN的稳态水平，但仅Mtb可耐受过量PepN的积累；与之相对，Msm则无法做到这一点。Msm不仅会对过量PepN（无论其为天然内源还是外源）进行蛋白水解，还会将其分泌至胞外。有趣的是，尽管PepN_Mtb是体内（in vivo）调控结核分枝杆菌毒力所必需的，而PepN_Msm却是耻垢分枝杆菌体外生长的必需基因。尽管二者的必需性存在显著差异，但PepN_Mtb与PepN_Msm均拥有几乎完全一致的N端M1型肽酶（M1-type peptidase）结构域：二者的氨基酸序列对齐度极高，二级结构也高度重合。而二者的C端内质网氨肽酶1 C端样（ERAP1_C-like）结构域的序列对齐度则相对较低。我们利用表达PepN_Msm的ΔpepN突变型Mtb开展了基于巨噬细胞的体外感染实验，结果显示PepN_Msm同样具备抵达宿主胞质的能力。最后值得关注的是，我们分别确定了PepN_Mtb与PepN_Msm的相互作用组（interactome），发现二者的重叠度极低：其中PepN_Mtb主要与Mtb的分泌蛋白发生相互作用，而PepN_Msm则主要与Msm的管家蛋白发生免疫共沉淀结合。综上，尽管两种PepN的序列同源性较高且具备多项共同特性，但本对比分析研究揭示：致病性分枝杆菌的PepN具有以宿主为中心的功能特性，而非致病性分枝杆菌的PepN则展现出以细菌自身为中心的功能特性。